Lymph transport occurs against a hydrostatic pressure gradient and thus relies critically on the intrinsic contractile function of lymphatic muscle, the ?active lymphatic pump?. Failure of this pump is associated with many types of lymphedema. Little is known about why lymphatic vessels become dysfunctional, but clinical studies reveal an elevated lymphatic diastolic pressure, enlarged diameter, impaired or absent contractions, and incompetent valves. These findings point to both pacemaker and valve dysfunction as underlying causes. This R01 renewal continues to address the ionic mechanisms of pacemaking in lymphatic vessels, with the ultimate goal of developing methods to treat pacemaker and contractile dysfunction in lymphedema. In the previous funding period genetic methods were used to assess the roles of multiple ion channels in mouse lymphatic smooth muscle and a critical role for Ano1 (TMEM16A) was found. SM-specific deletion resulted in a 3-4-fold reduction in pacemaking frequency and blunting or abolition of the increase in frequency in response to pressure elevation. This proposal continues to use tissue-specific and global mouse KO models to answer questions about ion channels that act in concert with Ano1 to initiate pacemaking in lymphatic smooth muscle cells (LMCs). It addresses not only how Ano1 is activated but also pressure-sensing mechanisms through mechanosensitive ion channels or G-protein-coupled receptors (GPCRs). Optogenetic tools will be used extensively for measuring intracellular Ca2+ events, uncaging Ca2+ or IP3 and triggering depolarization with channel rhodopsin. The central hypothesis is that a membrane oscillator generates a repetitive cycle of depolarization/repolarization to trigger lymphatic action potentials (APs) and this cycle is modulated by mechanosensitive ionic conductances and by G?q/11-mediated IP3 production / Ca2+ release. This hypothesis will be tested with 2 experimental aims and a numerical modeling aim to aid in the interpretation and integration of the underlying mechanisms: 1) Elucidate the Ca2+ sensitive ionic mechanisms that facilitate initiation of the lymphatic AP. Is another Ca2+-activated ion channel acting in combination with Ano1 to provide depolarization prior to AP firing? Is the slope of diastolic depolarization near the AP threshold determined by Ca2+ release events? Is a Ca2+-independent membrane oscillator involving HCN and Kv7 channels acting in combination with Ano1 as part of the pacemaking mechanism? 2) Determine the pressure- sensitive ionic mechanisms that regulate lymphatic pacemaking. Do mechanosensitive GPCRs coupled to G?q/11 drive IP3 production to regulate Ano1? Do mechanosensitive ion channels modulate depolarization or repolarization? In parallel, 3) Numerical models will be used to predict and verify ionic mechanisms that govern pacemaking, including the shape of the LMC action potential, the effect of pressure on diastolic depolarization, factors determining pacemaker initiation sites, and properties of rectifying myoendothelial junctions that might prevent current shunting to the endothelial layer but allow endothelial modulation of LMCs.